【作者】 张伟；

【导师】 刘俊秋；

【作者基本信息】 吉林大学 ， 高分子化学与物理， 2011， 博士

【摘要】 谷胱甘肽过氧化物酶(GPx)是机体内重要的抗氧化硒酶,具有保护机体免受氧化损伤功能。在分子及超分子水平上模拟含硒酶,无论对于研究酶催化机制,还是对于发展新型抗氧化药物,都具有十分重要的意义。本论文的研究思路立足于单分子含硒酶的分子设计,发展利用超分子自组装技术,构筑纳米酶自组装体,即从单分子蛋白质酶分子的结构改造入手,通过对两种蛋白质酶分子谷氧还蛋白(Grx)和脱氢抗坏血酸还原酶(DHAR)的活性中心进行重新设计和定向改造,获得了两种具有GPx催化功能的抗氧化含硒人工酶;在此基础上,以酶模型分子谷胱甘肽转硫酶(GST)为超分子构筑基元,通过金属配位相互作用,制备了具有高效催化功能的超分子酶自组装体,进一步讨论了单分子硒酶结构与功能的关系,以及“bottom up”纳米技术在酶催化功能材料设计中的应用:I基于人源谷氧还蛋白(hGrx1)的GPx仿酶设计:本研究工作发展了一种制备高效人源GPx人工酶的新方法。Grx具有同天然GPx相同的Trx折叠结构域和谷胱甘肽(GSH)底物识别特异性,由于该酶分子具有分子量小等优点,hGrx作为模拟GPx的天然蛋白骨架备受关注。本论文采用一种优化稀有密码子表达硒酶的新策略,制备了既能识别稀有密码子又能在外源蛋白氨基酸序列中插入硒代半胱氨酸(Sec)的表达菌株BL21cysE51 CodonPlus,成功制备了人源含硒酶Seleno-hGrx1;该硒酶Seleno-hGrx1展现出了高效的GPx催化活性,其催化活性可同天然GPx相媲美,稳态动力学分析表明,其催化行为属于典型的乒乓机制,与某些天然GPx相类似。该研究工作为设计药用抗氧化酶开拓了新思路。II基于脱氢抗坏血酸还原酶(DHAR)的GPx仿酶设计:利用蛋白超家族具有相同底物特异性的特点,成功将DHAR转化为人工GPx。DHAR和GPx同属Trx超家族,对底物GSH同样具有相似的底物识别特异性和亲和性。以DHAR为蛋白骨架,对DHAR活性中心的GSH结合位点进行了重新设计,利用Cys营养缺陷型表达技术引入GPx催化基团Sec,从而获得新型含硒人工酶。所制备的人工酶Seleno-DHAR展现了一定的GPx催化活性,同时该研究工作为深入探究天然GPx结构功能关系以及催化机制奠定了基础。III超分子酶自组装体的设计:发展了利用超分子自组装技术制备酶性纳米线的新方法。我们选用GST作为抗氧化酶模型蛋白,讨论了如何利用空间立体结构对称的同源二聚体GST为构筑基元,通过金属配位相互作用制备具有催化功能的纳米酶自组装体。即以GST为蛋白骨架,通过基因工程手段修饰分子识别配体His-tag,通过金属配位相互作用成功制备了形貌可控的线性酶纳米材料。论证了通过金属离子与His-tag的金属配位相互作用构筑蛋白质酶超分子纳米材料的可行性。在超分子水平上讨论抗氧化纳米酶模型的构建及催化行为,研究了单分子酶构筑基元与超分子酶纳米材料组装与解组装机制。该研究丰富了功能蛋白质超分子组装方法,为进一步设计具有抗氧化活性的多酶组装体奠定了基础。更多还原

【Abstract】 The formation of protein functional materials in natural biological systems occurs through self-assembly of protein building blocks whose structures are encoded by organism’s DNA. After transcription, translation and folding into functional building blocks in cells, the resultant individual protein components self-assemble at different size scales to form unique protein supramolecular structures with special physiological functions through molecular recognization, structural complementary and non-covalent interaction of fucntional protein building blocks in organisms. The dynamic biological assembling process of many natural protein superstructural materials involves a dynamic reversible change with physical and chemical conditions such as pH, temperature, and redox alterations.The knowledge of the details of the protein molecular recognition and supramolecular self-assembly mechanism in vivo enables us to mimic these processes in vitro through protein building block structural modification and functional redesign. The rational design of related supramolecular assemblies utilizing chemical compatibility and structural complementarity of individual protein modules based on the remarkable advance in protein engineering and biological supramolecular chemistry techniques should shed light on the principles for the development of‘bottom-up’nanotechnology and the design of functional protein nanomaterials with unique superstructures. In addition, it represents an important frontier of bionanotechnology with great potential for considerable contributions to biology, chemistry, as well as for the de novo design of biological materials with unique structures and functions.Based on the redesign of the active site and the molecular recognition mechanism of the supramolecular protein enzyme building blocks for the rational construction of potential functional biological nanomaterials, we redesigned the active site of human Grx1 and DHAR by incorporating catalytic amino acid residue Sec respectively to mimic the natural GPx catalytic function in vitro. The wild type human Grx1 and DHAR share a common substrate GSH binding site with natural GPx. In addition, we designed molecular recognition ligand of His-tag and introduced it to a C2 symmetric enzyme GST homodimer to prepare the bivalent supramolecular building block SjGST-6His, which could self-assemble to form linear enzyme nanomaterials with catalytic function through metal coordination between two different His-tags from two individual protein building blocks. At last, we discussed the principles for the redesign of the protein supramolecular building blocks in order to prepare functional biological nanomaterials and it should shed light on the the principles for the development of‘bottom-up’nanotechnology in the application of advanced functional protein nanomaterials.1. Seleno-hGrx1 with redesigned catalytic functionThe active center of human glutaredoxin (hGrx1) with excellent specific affinity for the substrate glutathione (GSH) was redesigned to introduce the catalytic selenocysteine residue for imitating the function of antioxidant selenoenzyme glutathione peroxidase (GPx) in vivo. The GSH-binding affinity of hGrx1 from human beings which shares a common thioredoxin fold with natural GPx makes it a competent candidate as antioxidant for potential medical application compared to other animal-originated protein scaffolds. Given that presence of two consecutive AGG-AGG rare codons for encoding Arg26-Arg27 residues in the reading frame of hGrx1 mRNA reduced Seleno-hGrx1 expression level significantly in the Cys auxotrophic E. coli strain BL21cysE51, a novel strategy for optimizing the rare codons was first reported and resulted in an remarkable increase of the expression level in the Cys auxotrophic cells for potential future medical production. The engineered artificial selenoenzyme displayed high GPx catalytic activity rivaling that of some natural GPx. The engineered Seleno-hGrx1 was characterized by kinetic analyses. It showed a typical ping-pong kinetic mechanism, and its catalytic properties were similar to those of some naturally occurring GPx.2. Seleno-DHAR with redesigned GPx functionDivergent evolution has resulted in superfamilies of enzymes with the same protein fold, but different catalytic performances. The disparate catalytic performances require different mechanistic steps, but some of these steps may be shared. The involved changing catalytic activity of an enzyme may just require a unique substitution of an amino acid residue at the active site. DHAR and GPx have distinct catalytic abilities, but share a common substrate GSH binding site and the same Trx fold like GST. DHAR was redesigned to incorporate catalytic Sec residue of GPx at the active site of DHAR in order to mimic the natural GPx catalytic ability by genetic engineering technique. The prepared selenium enzyme Seleno-DHAR displayed a remarkable GPx catalytic activity compared to wild type DHAR for GPx catalytic activity. And the strategy for converting DHAR catalytic activity to GPx catalytic activity is first reported and may shed light on the catalytic mechanism of natural GPx and the principles for the redesign of the function of enzyme supramolecular building blocks for rational design of the desired functional protein nanomaterials.3. Supramolecular self-assembly with catalytic functionA simple and versatile strategy for preparing functional enzyme nanowires based on self-assembly of a C2-symmetric homodimeric enzyme is proposed and demonstrated. The enzyme-loaded nanowires are fabricated throughout the outstretched His-tag recruiting a second building block by interprotein metal coordination. The prepared enzyme nanowires are characterized by tapping mode atom force microscopy. The height of one-dimensional nanowires forming at low protein concentration is about 4.0~5.0 nm, while nanowire height at the site of the entanglement crosslink of nanowires forming at higher protein concentration is about 7.0~9.0 nm, nearly twice the height of the single protein molecule (ca. 3.0~5.0 nm). The equilibrium of enzyme nanowires with individual protein blocks could be shifted and the geometric structure of the enzyme nanowires could be adjusted by modulating the concentration of the building blocks and metal ions. Given that the prepared enzyme nanowires introduce a minor difference in enzyme activity and second structure compared to individual building blocks, it is assumed that the metal-His tag coordination could enhance the enzyme nanowires thermostatic stability. The strategy may open a new avenue for designing large symmetrical materials in artificial control for a wide variety of proteins by adding His-tag to protein building blocks and shed light on the principles for the development of‘bottom-up’nanotechnology.更多还原